Project Summary Pseudomonas aeruginosa (Pa) is a major human pathogen responsible for extensive mortality and billions of dollars in health care costs. Pa infections are particularly devastating in Cystic Fibrosis (CF), a disease associated with defective ion transport, tenacious airway secretions, and chronic lung infections. Pa is particularly pathogenic in CF because of its ability to form robust biofilms - slimy conglomerates of polymers that allow Pa to colonize airways and to evade antibiotics. We recently identified Pf bacteriophages (phages) as a virulence factor that Pa uses to build biofilms. Pf phages are long, filamentous viruses produced by Pa that spontaneously organize extracellular matrix into a liquid crystal. This assembly is driven by entropic, charge-based interactions between Pf phages and host and microbial polymers. The resulting crystalline architecture fortifies biofilms, making them more viscous, adherent, and tolerant to antibiotics. We recently reported that many CF patients have crystalline sputum. Because Pf phage-mediated polymer crystallization is highly concentration-dependent, sputum Pf phage titers in CF patients may have clinical significance. However, the prevalence and clinical impact of Pf phages in CF patients are unknown. Along with enhancing the pathogenic features of established Pa biofilms, Pf phage may also promote the initial establishment of Pa infections. Pa dramatically increase Pf phage production without hours of intra-tracheal instillation in a mouse model of acute lung infection. Our preliminary data indicate that mice in this model clear Pa strains lacking Pf phage while Pf phage-competent strains reliably establish infections. In light of these contributions to the pathogenesis of Pa lung infections, we propose that Pa may partner symbiotically with Pf phage to promote Pa lung infections. It may be advantageous to target Pf phage therapeutically. To this end, we have developed anti-Pf phage vaccines and monoclonal antibodies that target Pf phage and disrupt crystallization. Our data suggest that active or passive immunization against Pf phage may be an effective strategy for preventing Pa infection. In light of these exciting preliminary data, we propose to test the hypothesis that Pf phages are critical virulence factors that can be targeted to prevent chronic Pa infections. To test this, In Aim 1 we will characterize how Pf phages impact clinical outcomes in CF. In Aim 2 we will define how anti-Pf phage antibody levels influence clinical outcomes in CF. Finally, in Aim 3 we will determine whether anti-Pf phage vaccines and monoclonal antibodies prevent the establishment of Pa infections in animal models. Together, these aims represent a bold, imaginative, and radically unconventional approach to Pa biofilm Infections. If successful, this work will provide the clinical rationale and mechanistic foundation needed to support R01-level investigations into the efficacy of anti-Pf phage immunization in CF and other settings.